Scaffolded fusion polypeptides and compositions and methods for making the same

Abstract

The present invention provides soluble forms of integral membrane proteins, or domains or portions thereof, that retain the biological activity of the integral membrane protein, domain or portion from which they are designed or derived and that can readily be expressed in high yield.

Description

1. FIELD OF THE INVENTIONThe present invention relates to novel compositions designed from integral membrane proteins that have improved solubility and retain biological activity.2. BACKGROUND OF THE INVENTIONMany important cellular and biological processes are mediated by integral membrane proteins. Often quantities of these integral membrane proteins are required to study their roles in the critical cellular and biological processes. However, integral membrane proteins are often difficult to obtain in quantity while retaining solubility and function.For example, according to one common technique, the integral membrane protein is extracted from the bilayer with a nonionic detergent. Unfortunately, the proteins recovered by such extraction techniques frequently lack activity.In another common technique, a soluble portion of the membrane protein, such as an extracellular domain, is expressed in the absence of the insoluble portions of the membrane protein (typically the transmembrane...

AI Technical Summary

Benefits of technology

Taken together, the scaffold strands of the scaffolded fusion polypeptide constitute a scaffold domain. The scaffold domain mimics the TMD adjacent the ECD or ICD loop of the integral membrane protein from which the scaffolded fusion polypeptide of the invention is designed, with one significant difference. Whereas the TMDs of integral membrane proteins are hydrophobic in nature, thereby causing the integral membrane proteins to be insoluble in membrane-free and detergent-free aqueous medium, the scaffold domains of the scaffolded fusion polypeptides of the invention display improved solubility in such aqueous solutions.

A significant advantage of the scaffolded fusion polypeptides of the invention is that they are modular structures that can be linked together to form polymeric scaffolded fusion polypeptides. One module of such a polymeric scaffolded fusion polypeptide comprises a functional domain and a scaffold domain, as described above. A scaffolded fusion polypeptide can comprise a plurality of these modules ("polymeric scaffolded fusion polypeptides"), which permits the design and synthesis of forms of integral membrane proteins that span the membrane multiple times, such as, for example, soluble forms of cytokine receptors, G-protein coupled receptors (GCPR), ion channel receptors, and other integral membrane proteins known to those of skill in the art. These forms of the integral membrane proteins display significantly improved solubility. One exemplary polymeric scaffolded fusion polypeptide is a soluble form of the ECD of the CCR5 transmembrane receptor implicated in HIV infection.

In the polymeric forms of the scaffolded fusion polypeptides of the invention, the modules are linked together via their scaffold strands, either directly or with an optional linker or spacer. For example, the second scaffold strand of the first module may be linked to the first scaffold strand of the second module, and so forth. The functional domains of a polymeric scaffolded fusion protein can all correspond to regions of a single protein of interest, or they can correspond to regions from different proteins of interest. For example, the functional domain of the first module can correspond to a loop of the ECD of a first GPCR and the functional domain of the second module can correspond to a loop of the ECD of a second, different GPCR. As another example, the functional domains of a polymeric scaffolded fusion polypeptide can each correspond to soluble loops of two different transmembrane receptors that form a complex in the membrane. This modular format provides great flexibility regarding the types and complexities of the scaffolded fusion polypeptides that can be created.

Problems solved by technology

However, integral membrane proteins are often difficult to obtain in quantity while retaining solubility and function.

Unfortunately, the proteins recovered by such extraction techniques frequently lack activity.

However, this method is inadequate for expressing the soluble portions of many membrane proteins, especially those that traverse the membrane more than once.

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